Cryptosporidium parvum (phylum Apicomplexa) is a coccidian protozoan capable of parasitizing the intestinal tract of a variety of mammalian species. The protozoan is generally believed to be spread by an oral-fecal route, infecting the intestinal epithelia and, to a lesser extent, the extraintestinal epithelia causing severe diarrhea. C. parvum has been increasingly recognized as the causative agent of recent waterborne outbreaks of gastroenteritis.
Although usually self-limiting, the diarrheal disease can be prolonged and life threatening to the young and to the immunosuppressed. While first recognized as a veterinary pathogen, cryptosporidiosis has gained in importance with the spread of AIDS-related immunosuppression. Further, numerous outbreaks of cryptosporidiosis have been reported in the United States involving children attending day-care facilities (Jenkins et al., 1997, U.S. Pat. No. 5,591,434, which is incorporated by reference herein as if set forth in its entirety). The disease has also been implicated as having a significant role in the vicious cycle of malnutrition/diarrhea among children in developing countries.
In a recent study, the 50% infectious dose in normal immunocompetent individuals was determined to be 132 oocysts and as few as 30 oocysts caused infection (Dupont et al., 1995, N. Eng. J. Med. 332:855-859). At present, there does not appear to be any prophylactic therapy available to prevent this parasitic disease in humans or in animals (Jenkins et al., supra), and there is no effective chemotherapeutic treatment for cryptosporidiosis once the host has been infected.
Cryptosporidium parvum exists in nature in the form of environmentally resistant, thick-walled oocysts. The oocysts are known to remain viable for extended periods of time and are resistant to conventional water disinfection methods. Due to massive shedding of oocysts in the feces of infected animals or individuals and the robust nature of the oocysts, they are frequently present in raw surface water (LeChevallier et al., 1991, Appl. Environ. Microbiol. 56:1423-1428) and finished drinking water (LeChevallier and Norton, 1995, J. Am. Water Works Assoc. 87:54-68). Currently, the U.S. Environmental Protection Agency Information Collection Rule (ICR) mandates the use of the indirect fluorescent antibody (IFA) method for the detection of Cryptosporidium oocysts in water concentrates (U.S. Environmental Protection Agency, 1996, Fed. Regist. 61:24354-24388). While the IFA method does not distinguish between viable and nonviable oocysts, the use of fluorogenic vital dyes may distinguish between viable and nonviable oocysts (Campbell et al., 1992, Appl. Environ. Microbiol. 58:3488-3493). However, the use of these microscopic techniques is hampered by their labor-intensive, time-consuming nature, their inability to distinguish between human pathogenic C. parvum oocyst and the oocyst of animal pathogenic Cryptosporidium species, and the inability of the tests to differentiate infectious and non-infectious oocysts.
Oocyst resistance to chlorination, difficulties in effective methods for detection of the parasite, and a lack of effective treatment for cryptosporidiosis have contributed to the spread of the organism. Accordingly, the importance of prophylaxis by detection of the parasite in drinking water before it is ingested by the human host has given impetus to the development of methods for the effective detection of C. parvum in water samples.
Recently, various strategies have been combined with standard polymerase chain reaction assay (PCR) to detect the presence of viable C. parvum oocysts, including integration of in vivo excystation (Deng et al., 1997, Appl. Environ. Microbiol. 63:3134-3138; Filkhorn et al., 1994, Zentralbl. Hyg. Unweltmed. 195:489-494; Wagner-Weining and Kimmig, 1995, Appl. Environ. Microbiol. 61:4514-4516), and the use of reverse-transcriptase PCR (RT-PCR) for the detection of mRNA transcripts found only in viable oocysts (Rochelle et al., 1997, Appl. Environ. Microbiol. 63:2029-2037; Stinear et al., 1996, Appl. Environ. Microbiol. 62:3385-3390). Further, infectivity assessment by integrated cell culture-PCR (CC-PCR) has also been developed (Di Giovanni et al., 1997, Proceedings of the American Water Works Assoc., Water Quality Tech. Conf., Denver, CO; De Leon and Rochelle., 1998, U.S. Pat. No. 5,770,368; Rochelle et al., 1997, Appl. Environ. Microbiol. 63:2029-2037). However, although these prior art methods provide high specificity and sensitivity, they do not provide a quantitative measure of starting target nucleic acid copy numbers and, therefore, the prior art methods do not accurately measure the numbers of oocysts in the samples. Further, until the present invention, there is no report that CC-PCR has been successfully used to detect infectious C. parvum in environmental raw water samples.
Although there has been limited success in detection of oocysts in environmental raw water samples using PCR methods, CC-PCR detection of infectious C. parvum oocysts in environmental raw water samples has been hampered by, among other things, difficulties in: removing inhibitors of PCR, quantitation of organisms, ensuring that sufficient equivalent volume is assayed, removal of compounds cytotoxic to the cell culture, and ensuring that the process of collection and analysis does not inactivate the oocysts. Nonetheless, the detection of infectious C. parvum oocyst contamination in environmental raw water samples is important in raw-source water assessment for effective watershed management and also in enabling the evaluation of the efficiency and quality of drinking water treatment procedures. Therefore, while the methods of present invention can be used to detect infectious oocysts in finished drinking water to protect the public health, the procedures disclosed herein also permit the examination of, inter alia, raw water, process water, and waste streams which is of great value for understanding parasite occurrence and the efficacy of water treatment processes.
Further, the prior art PCR-based assays for C. parvum require confirmation that the parasite is infectious and/or that the DNA amplified is, indeed, the C. parvum-specific target sequence by complex methodologies requiring, inter alia, skilled persons trained in recombinant DNA techniques. For example, heat shocking the organisms followed by reverse transcriptase PCR (RT-PCR) has been used to detect viable organisms (De Leon and Rochelle, 1998, U.S. Pat. No. 5,770,368) (hereinafter the ""368 patent). That patent also teaches the alternative procedure of inoculating susceptible mammalian cells with a sample in order to detect infectious C. parvum by RT-PGR.
In either case, all PCR-based prior art methods require that the PCR amplification products be examined using recombinant DNA techniques to confirm that the C. parvum target nucleic acid sequence has, indeed, been specifically amplified. Typically, PCR amplification products are separated according to size on agarose gels and then the fragments are visualized by ethidium bromide-fluorescence staining under ultraviolet light to determine the presence of the appropriately-sized fragment as predicted by the primer pair used to amplify the target nucleic acid sequence. The actual sequence identity of the putative PCR fragment may be confirmed by Southern blot hybridization using a short internal DNA probe which complements the DNA expected in the PCR product and which is labeled so that it may be detected on the blot. Thus, when the target PCR product has been generated by the amplification reaction, the matching labeled probe will hybridize to it thereby confirming the product""s identity. Alternatively, the putative PCR product may be cloned and sequenced by standard methods. Thus, the prior art methods require complex, time-consuming sample handling by a technician skilled in recombinant DNA methodology.
Moreover, none of the prior art PCR-based methods are quantitative and, therefore, those methods do not provide a measure of the contamination level of the water sample assayed. This is because PCR, although highly sensitive, does not measure the initial amount of target nucleic acid in a sample. Indeed, prior art assays which identify viable organisms in a sample require additional steps preceding PCR amplification, i.e., heat shocking and reverse transcriptase, which further hinder quantitation as well as increase the opportunity for operator error in the detection procedure. Further, heat shocking followed by RT-PCR may provide some information on viability but does not assess the infectivity of the oocysts. Additionally, RT-PCR requires a much longer processing time, increases the complexity and cost of the detection procedure, and may be hampered by interference from RNases present in the sample which may degrade the mRNA product thereby producing false negative results and allowing the parasite to go undetected. Therefore, there is a significant need in the art for a simple, efficient, and preferably quantitative method for detecting and/or quantifying this potentially lethal pathogen in water samples.
The invention relates to the specific and, preferably, quantitative detection of C. parvum oocysts in aqueous samples.
The invention includes a method useful for detecting a Cryptosporidium parvum organism m an aqueous sample. The method comprises the steps of
(a) concentrating any Cryptosporidium parvum organism present in the sample by immunomagnetic separation;
(b) amplifying a target Cryptosporidium parvum nucleic acid present in the sample after step (a);
(c) detecting any amplified target nucleic acid formed in step (b) to detect a Cryptosporidium parvum organism in an aqueous sample.
In one aspect, the aqueous sample is selected from the group consisting of an environmental raw water sample, a backwash water sample, a process water sample, and a finished water sample.
In another aspect, the method further comprises infecting a susceptible mammalian cell culture with Cryptosporidium parvum organisms concentrated in step (a), and producing the Cryptosporidium parvum target nucleic acid prior to amplifying the target nucleic acid in step (b).
In preferred embodiments, the mammalian cell culture is selected from the group consisting of a HCT-8 cell culture, a CaCo-2 cell culture, and a MDBK cell culture.
In another aspect, the cell culture is incubated for a period of time from about 4 hours to about 72 hours following inoculation.
In yet another aspect, step (b) further comprises hybridizing any amplified target nucleic acid formed with a fluorescer-quencher oligonucleotide probe specific for said target Cryptosporidium parvum nucleic acid and measuring the level of fluorescence in the sample, wherein the level of fluorescence is quantitatively correlated to the amount of Cryptosporidium parvum oocysts in the sample.
In another aspect, the fluorescer molecule is attached to the 5xe2x80x2 end of the oligonucleotide probe, and further wherein the fluorescer molecule is selected from the group consisting of a 6-carboxyfluorescein fluorescer molecule, a tetrachloro-6-carboxyfluorescein fluorescer molecule, a 2,7,-dimethoxy-4,5-dichloro-6-carboxyfluorescein fluorescer molecule, and a hexachloro-6-carboxyfluorescein fluorescer molecule.
In yet another aspect, the quencher molecule 6-carboxytetramethylrhodamine is attached to the 3xe2x80x2 end of the oligonucleotide probe.
A method useful for quantitatively detecting a Cryptosporidium parvum organism in a sample is also included in the invention. The method comprises the steps of
(a) concentrating any Cryptosporidium parvum organism in the sample by immunomagnetic separation;
(b) amplifying a target Cryptosporidium parvum nucleic acid;
(c) hybridizing any amplified target nucleic acid formed in step (b) with a fluorescer-quencher oligonucleotide probe specific for the target Cryptosporidium parvum nucleic acid; and
(d) detecting any amplified target nucleic acid by quantitative sequence detection, wherein the level of fluorescence is correlated to the quantitative measure of the amount of Cryptosporidium parvum oocysts in the sample, to quantitatively detect a Cryptosporidium parvum organism in a sample.
In one aspect, the nucleic acid is DNA.
The invention also includes a method of quantitatively detecting a Cryptosporidium parvum organism in an aqueous sample. The method comprises the steps of
(a) contacting a target Cryptosporidium parvum nucleic acid with polymerase chain reaction reagents specific for the target nucleic acid, the polymerase chain reaction reagents including at least two polymerase chain reaction primers, a polymerase enzyme, and an oligonucleotide probe, the oligonucleotide probe further comprising:
(i) a sequence capable of hybridizing to a portion of the target Cryptosporidium parvum nucleic acid wherein the portion is unique to Cryptosporidium parvum; 
(ii) a fluorescer molecule attached to a first end of the oligonucleotide;
(iii) a quencher molecule attached to a second end of said oligonucleotide capable of substantially quenching the fluorescer molecule when the oligonucleotide is intact, wherein the fluorescer molecule becomes substantially unquenched when the oligonucleotide probe is cleaved by DNA polymerase during amplification of the Cryptosporidium parvum target nucleic acid; and
(iv) a 3xe2x80x2 end which is impervious to the 5xe2x80x2xe2x86x923xe2x80x2 extension activity of the DNA polymerase; and
(b) amplifying the target Cryptosporidium parvum nucleic acid by thermal cycling, wherein the thermal cycling is sufficient to amplify the target nucleic acid; and
(c) measuring the level of fluorescence in the sample subsequent to thermal cycling, and further wherein the level of fluorescence is correlated to the amount of Cryptosporidium parvum oocysts present in the sample, to quantitatively detect a Cryptosporidium parvum organism in an aqueous sample.
In preferred embodiments, the aqueous sample is selected from the group consisting of an environmental raw water sample, a backwash water sample, a process water sample, and a finished water sample.
In one aspect, the method further comprises concentrating any Cryptosporidium parvum organism present in the sample by immunomagnetic separation prior to step (a).
In another aspect, the method further comprises infecting a susceptible mammalian cell culture with any Cryptosporidium parvum organism concentrated by immunomagnetic separation and producing the Cryptosporidium parvum target nucleic acid prior to step (a).
The invention also includes a kit for detecting a C. parvum nucleic acid, the kit comprising a first nucleic acid having the sequence SEQ ID NO:2, and a second nucleic acid having the sequence SEQ ID NO:3.
In one aspect, the kit comprises an oligonucleotide probe complementary to at least a portion of a nucleic acid having the sequence of residues 1120-1465 of SEQ ID NO:1.
In another aspect, the oligonucleotide probe has the sequence SEQ ID NO:4.